Influenza A(H3N2) viruses evade human immunity primarily by acquiring antigenic changes in the haemagglutinin (HA). HA receptor-binding features of contemporary A(H3N2) viruses hinder traditional antigenic characterization using haemagglutination inhibition and promote selection of HA mutants. Thus, alternative approaches are needed to reliably assess antigenic relatedness between circulating viruses and vaccines. We developed a high content imaging-based neutralization test (HINT) to reduce antigenic mischaracterization resulting from virus adaptation to cell culture. Ferret reference antisera were raised using clinical specimens containing viruses representing recent vaccine strains. Analysis of viruses circulating during 2011–2018 showed that gain of an N158-linked glycosylation in HA was a molecular determinant of antigenic distancing between A/Hong Kong/4801/2014-like (clade 3C.2a) and A/Texas/50/2012-like viruses (clade 3C.1), while multiple evolutionary HA F193S substitution were linked to antigenic distancing from A/Switzerland/97152963/2013-like (clade 3C.3a) and further antigenic distancing from A/Texas/50/2012-like viruses. Additionally, a few viruses carrying HA T135K and/or I192T showed reduced neutralization by A/Hong Kong/4801/2014-like antiserum. Notably, this technique elucidated the antigenic characteristics of clinical specimens, enabling direct characterization of viruses produced in vivo , and eliminating in vitro culture, which rapidly alters the genotype/phenotype. HINT is a valuable new antigenic analysis tool for vaccine strain selection.

West Nile virus (WNV) was introduced to New York in 1999 and rapidly spread throughout North America and into parts of Central and South America. Displacement of the original New York (NY99) genotype by the North America/West Nile 2002 (NA/WN02) genotype occurred in 2002 with subsequent identification of a novel genotype in 2003 in isolates collected from the southwestern Unites States region (SW/WN03 genotype). Both genotypes co-circulate to date. Subsequent WNV surveillance studies have confirmed additional genotypes in the United States that have become extinct due to lack of a selective advantage or stochastic effect; however, the dynamic emergence, displacement, and extinction of multiple WNV genotypes in the US from 1999–2012 indicates the continued evolution of WNV in North America.

West Nile virus (WNV) has been maintained in North America in enzootic cycles between mosquitoes and birds since it was first described in North America in 1999. House sparrows (HOSPs; Passer domesticus) are a highly competent host for WNV that have contributed to the rapid spread of WNV across the U.S.; however, their competence has been evaluated primarily using an early WNV strain (NY99) that is no longer circulating. Herein, we report that the competence of wild HOSPs for the NY99 strain has decreased significantly over time, suggesting that HOSPs may have developed resistance to this early WNV strain. Moreover, recently isolated WNV strains generate higher peak viremias and mortality in contemporary HOSPs compared to NY99. These data indicate that opposing selective pressures in both the virus and avian host have resulted in a net increase in the level of host competence of North American HOSPs for currently circulating WNV strains.

Background: PKR regulates many biological processes including stress response. Results: An activation-loop-phosphorylation bypass mutant of PKR functions without a key dimer-interface residue. The PKR kinase domain together with a kinase-dead partner, but not alone, activates the catalytic function. Conclusion: Activation loop phosphorylation occurs in cis following dimerization. Significance: PKR autophosphorylates in the activation loop in cis to initiate the antiviral signaling cascade.